Min-Jeong Song

Endoplasmic Reticulum Stress-Induced JNK Activation Is a Critical Event Leading to Mitochondria-Mediated Cell Death Caused by β-Lapachone Treatment

Background β-lapachone (β-lap) is a bioreductive agent that is activated by the two-electron reductase NAD(P)H quinone oxidoreductase 1 (NQO1). Although β-lap has been reported to induce apoptosis in various cancer types in an NQO1-dependent manner, the signaling pathways by which β-lap causes apoptosis are poorly understood. Methodology/Principal Findings β-lap-induced apoptosis and related molecular signaling pathways in NQO1-negative and NQO1-overexpressing MDA-MB-231 cells were investigated. Pharmacological inhibitors or siRNAs against factors involved in β-lap-induced apoptosis were used to clarify the roles played by such factors in β-lap-activated apoptotic signaling pathways. β-lap leads to clonogenic cell death and apoptosis in an NQO1- dependent manner. Treatment of NQO1-overexpressing MDA-MB-231 cells with β-lap causes rapid disruption of mitochondrial membrane potential, nuclear translocation of AIF and Endo G from mitochondria, and subsequent caspase-independent apoptotic cell death. siRNAs targeting AIF and Endo G effectively attenuate β-lap-induced clonogenic and apoptotic cell death. Moreover, β-lap induces cleavage of Bax, which accumulates in mitochondria, coinciding with the observed changes in mitochondria membrane potential. Pretreatment with Salubrinal (Sal), an endoplasmic reticulum (ER) stress inhibitor, efficiently attenuates JNK activation caused by β-lap, and subsequent mitochondria-mediated cell death. In addition, β-lap-induced generation and mitochondrial translocation of cleaved Bax are efficiently blocked by JNK inhibition. Conclusions/Significance Our results indicate that β-lap triggers induction of endoplasmic reticulum (ER) stress, thereby leading to JNK activation and mitochondria-mediated apoptosis. The signaling pathways that we revealed in this study may significantly contribute to an improvement of NQO1-directed tumor therapies.

β-Lapachone Significantly Increases the Effect of Ionizing Radiation to Cause Mitochondrial Apoptosis via JNK Activation in Cancer Cells

Background β-lapachone (β-lap), has been known to cause NQO1-dependnet death in cancer cells and sensitize cancer cells to ionizing radiation (IR). We investigated the mechanisms underlying the radiosensitization caused by β-lap. Methodology/Principal Findings β-lap enhanced the effect of IR to cause clonogenic cells in NQO1+-MDA-MB-231 cells but not in NQO1−-MDA-MB-231 cells. β-lap caused apoptosis only in NQO1+ cells and not in NQO1− cells and it markedly increased IR-induced apoptosis only in NQO1+ cells. Combined treatment of NQO1+ cells induced ROS generation, triggered ER stress and stimulated activation of ERK and JNK. Inhibition of ROS generation by NAC effectively attenuated the activation of ERK and JNK, induction of ER stress, and subsequent apoptosis. Importantly, inhibition of ERK abolished ROS generation and ER stress, whereas inhibition of JNK did not, indicating that positive feedback regulation between ERK activation and ROS generation triggers ER stress in response to combined treatment. Furthermore, prevention of ER stress completely blocked combination treatment-induced JNK activation and subsequent apoptotic cell death. In addition, combined treatment efficiently induced the mitochondrial translocation of cleaved Bax, disrupted mitochondrial membrane potential, and the nuclear translocation of AIF, all of which were efficiently blocked by a JNK inhibitor. Caspases 3, 8 and 9 were activated by combined treatment but inhibition of these caspases did not abolish apoptosis indicating caspase activation played a minor role in the induction of apoptosis. Conclusions/Significance β-lap causes NQO1-dependent radiosensitization of cancer cells. When NQO1+ cells are treated with combination of IR and β-lap, positive feedback regulation between ERK and ROS leads to ER stress causing JNK activation and mitochondrial translocation of cleaved Bax. The resultant decrease in mitochondrial membrane leads to translocation of AIF and apoptosis.

Radiation-induced angiogenic signaling pathway in endothelial cells obtained from normal and cancer tissue of human breast

Despite strong possibility that endothelial cells (ECs) of tumors and normal tissues may differ in various aspects, most previous studies on ECs have used normal cells. Here, we purified ECs from tumorous and normal human breast tissues, and studied the effect of radiation on angiogenesis and relevant molecular mechanisms in these cells. We found that in normal tissue-derived ECs (NECs), 4 Gy irradiation increased tube formation, matrix metalloproteinase 2 (MMP-2) expression and extracellular signal-regulated kinase (ERK) pathway activation. In cancer-derived ECs (CECs), however, 4 Gy irradiation significantly reduced tube formation, increased the production of angiostatin and interleukin-6 (IL-6), and upregulated AKT and c-Jun N-terminal kinase (JNK) pathway activation. Knockdown experiments showed that siMMP-2 efficiently inhibited tube formation by irradiated NECs, whereas siPlasminogen effectively attenuated the radiation-induced suppression of tube formation and the upregulation of angiostatin in CECs. Moreover, siIL-6 clearly inhibited the radiation-induced generation of angiostatin in CECs. Inhibition of ERK with a pharmacological inhibitor or small interfering RNAs (siRNAs) markedly suppressed the radiation-induced tube formation and MMP-2 upregulation in NECs, whereas the inhibition of either AKT or JNK with pharmacological inhibitor or siRNA treatment of CECs markedly attenuated the inhibition of tube formation and the upregulation of angiostatin and IL-6 caused by 4 Gy irradiation. These observations collectively demonstrate that there are distinct differences in the radiation responses of NECs and CECs, and might provide important clues for improving the efficacy of radiation therapy.

Radio-sensitivities and angiogenic signaling pathways of irradiated normal endothelial cells derived from diverse human organs

The purpose of the present investigation was to study the effects of ionizing radiation on endothelial cells derived from diverse normal tissues. We first compared the effects of radiation on clonogenic survival and tube formation of endothelial cells, and then investigated the molecular signaling pathways involved in endothelial cell survival and angiogenesis. Among the different endothelial cells studied, human hepatic sinusoidal endothelial cells (HHSECs) were the most radio-resistant and human dermal microvascular endothelial cells were the most radio-sensitive. The radio-resistance of HHSECs was related to adenosine monophosphate-activated protein kinase and p38 mitogen-activated protein kinase-mediated expression of MMP-2 and VEGFR-2, whereas the increased radio-sensitivity of HDMECs was related to extracellular signal-regulated kinase-mediated generation of angiostatin. These observations demonstrate that there are distinct differences in the radiation responses of normal endothelial cells obtained from diverse organs, which may provide important clues for protection of normal tissue from radiation exposure.

The anti-tumour compound, RH1, causes mitochondria-mediated apoptosis by activating c-Jun N-terminal kinase

BACKGROUND AND PURPOSE 2,5‐diaziridinyl‐3‐(hydroxymethyl)‐6‐methyl‐1,4‐benzoquinone (RH1) is a bioreductive agent that is activated by the two‐electron reductase NAD(P)H quinone oxidoreductase 1 (NQO1). Although the cytotoxic efficacy of RH1 against tumours has been studied extensively, the molecular mechanisms underlying this anti‐cancer activity have not yet been fully elucidated.

The radiosensitivity of endothelial cells isolated from human breast cancer and normal tissue in vitro

We developed a novel method for harvesting endothelial cells from blood vessels of freshly obtained cancer and adjacent normal tissue of human breast, and compared the response of the cancer-derived endothelial cells (CECs) and normal tissue-derived endothelial cells (NECs) to ionizing radiation. In brief, when tissues were embedded in Matrigel and cultured in endothelial cell culture medium (ECM) containing growth factors, endothelial cells grew out of the tissues. The endothelial cells were harvested and cultured as monolayer cells in plates coated with gelatin, and the cells of 2nd-5th passages were used for experiments. Both CECs and NECs expressed almost the same levels of surface markers CD31, CD105 and TEM-8 (tumor endothelial marker-8), which are known to be expressed in angiogenic endothelial cells, i.e., mitotically active endothelial cells. Furthermore, both CECs and NECs were able to migrate into experimental wound in the monolayer culture, and also to form capillary-like tubes on Matrigel-coated plates. However, the radiation-induced suppressions of migration and capillary-like tube formations were greater for CECs than NECs from the same patients. In addition, in vitro clonogenic survival assays demonstrated that CECs were far more radiosensitive than NECs. In summary, we have developed a simple and efficient new method for isolating endothelial cells from cancer and normal tissue, and demonstrated for the first time that endothelial cells of human breast cancer are significantly more radiosensitive than their normal counterparts from the same patients.

NQO1 prevents radiation-induced aneuploidy by interacting with Aurora-A.

Aneuploidy is the most common characteristic of human cancer cells. It also causes genomic instability, which is involved in the initiation of cancer development. Various lines of evidence indicate that nicotinamide adenine dinucleotide(P)H quinone oxidoreductase 1 (NQO1) plays an important role in cancer prevention, but the molecular mechanisms underlying this effect have not yet been fully elucidated. Here, we report that ionizing radiation (IR) induces substantial aneuploidy and centrosome amplification in NQO1-deficient cancer cells, suggesting that NQO1 plays a crucial role in preventing aneuploidy. NQO1 deficiency markedly increased the protein stability of Aurora-A in irradiated cancer cells. Small interfering RNA targeting Aurora-A effectively attenuated IR-induced centrosome amplification concerned with aneuploidy in NQO1-deficient cancer cells. Furthermore, we found that NQO1 specifically binds to Aurora-A via competing with the microtubule-binding protein, TPX2 (targeting protein for Xklp2), and contributes to the degradation of Aurora-A. Our results collectively demonstrate that NQO1 plays a key role in suppressing IR-induced centrosome amplification and aneuploidy through a direct interaction with Aurora-A.

Abstract A11: Radio-sensitivities and angiogenic signaling pathway of irradiated endothelial cells isolated from cancer and normal tissue of human breasts in vitro

Despite the strong possibility that endothelial cells of tumors and normal tissues may be different in various aspects, most of the previous studies on endothelial cells have been conducted using normal endothelial cells. Therefore, we have developed a novel method for harvesting endothelial cells from blood vessels of freshly obtained cancer and adjacent normal tissue of human breast, and further investigated radio-sensitivities and angiogenic signaling pathway in the alternation of angiogenic process observed after the purified cancer-derived endothelial cells (CECs) and normal tissue-derived endothelial cells (NECs) were irradiated in vitro. When human breast tissues including cancer were embedded in Matrigel and cultured in endothelial cell culture medium (ECM) containing growth factors, endothelial cells grew out of the tissues. The endothelial cells were harvested, cultured as monolayer cells in plates coated with gelatin, and exposed to radiation, and then the changes in clonogenic cell survival, tube formation capacity, γH2AX foci, gene expression, and cellular signaling pathways were determined. Both CECs and NECs expressed almost the same levels of surface markers CD31, CD105 and TEM-8 (tumor endothelial marker-8), which are known to be expressed in angiogenic endothelial cells, i.e., mitotically active endothelial cells. CECs were significantly more radiosensitive in treatment of single dose (4 Gy) and hyperfractionation (4 Gy in 2 or 4 equal fractions) of ionizing radiation (IR) than their normal counterparts although hyperfractionated IR reduced clonogenic cell death in both NECs and CECs than single dose of IR. In addition, we found that IR-induced foci formation of γH2AX in NECs increased 1 hr and declined afterwards when the cells were treated with 4 Gy of IR. On the contrary, it was remained up to 4 hr in CECs. In NECs, 4 Gy of IR induced an increase in the capillary-like tube formation, the expression of matrix metalloproteinase-2 (MMP-2), and the activation of ERK pathway. However, in CECs, 4 Gy of IR significantly reduced the capillary-like tube formation, and induced the expression of angiostatin and the activation of both AKT and JNK pathways. Additionally, inhibition of ERK with a pharmacological inhibitor or a small interfering RNA markedly suppressed the capillary-like tube formation and the expression of MMP-2 caused by IR in NECs. In CECs, inhibition of either AKT or JNK with a pharmacological inhibitor or a small interfering RNA clearly attenuated inhibition of the capillary-like tube formation, and the expression of angiostatin caused by IR. Furthermore, hypoxia increased the capillary-like tube formation and radioresistance in both NECs and CECs, but the delineation of the mechanisms remains to be studied. We have developed a simple and efficient new method for isolating endothelial cells from cancer and normal tissue. Moreover, our results collectively demonstrate that there are distinct differences in the radiation responses of NECs and CECs, and might provide important clues for improving the efficacy of radiation therapy. [This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (MRC: NRF-2014R1A5A2009392 and NRF-2013M2A2A7043703] Citation Format: Eun-Taex Oh, Min-Jeong Song, Hyemi Lee, Yun-Jeong Choi, Heon Joo Park. Radio-sensitivities and angiogenic signaling pathway of irradiated endothelial cells isolated from cancer and normal tissue of human breasts in vitro. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A11.

Abstract 409: Docetaxel induced-JNK2/PHD1 signaling pathway increases cell death in cancer cells under hypoxia through the degradation of HIF-1α

HIF-1α has been shown to regulate the expression of more than 70 genes involved in angiogenesis, tumor growth, metastasis, chemoresistance and radioresistance. There has been a growing interest in using HIF-1α inhibitors as anticancer drugs. Recently, docetaxel, which has been approved by the FDA for treatment of various cancers, has been reported to enhance the degradation of HIF-1α, but its molecular mechanism is unclear. In this study, we investigated the molecular mechanism underlying the docetaxel-induced degradation of HIF-1α and cell death in cancer cell under hypoxia. Pretreatment of docetaxel increased the polyubiquitination and proteasome mediated-degradation of HIF-1α and cell death in cancer cells under hypoxia. Under hypoxia, pharmacological inhibitors or siRNAs for PHD1 (prolyl hydroxylase 1) effectively prevented HIF-1α degradation and cancer cell death through inhibiting docetaxel-mediated activation of PHD1. In addition, siRNA-mediated knockdown of JNK2 blocked docetaxel-induced degradation of HIF-1α and cancer cell death through inhibiting the activation of PHD1. Through a luciferase reporter assay, we observed that the inhibition of the JNK2/PHD1 signaling pathway significantly suppressed the transcriptional activity of HIF-1α. Our results collectively show that, under hypoxia, docetaxel contributes to cause apoptotic cell death under hypoxia by triggering the JNK2/PHD1 signaling pathway, thereby increasing the degradation of HIF-1α. Citation Format: Eun-Taex Oh, Min-Jeong Song, Hyemi Lee, Yun-Jeong Choi, Heon Joo Park. Docetaxel induced-JNK2/PHD1 signaling pathway increases cell death in cancer cells under hypoxia through the degradation of HIF-1α. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 409. doi:10.1158/1538-7445.AM2015-409

Abstract 3905: Radiation-induced angiogenic signaling pathway in endothelial cells isolated from normal and cancer tissue of human breast.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Despite the strong possibility that the endothelial cells (ECs) of tumors and normal tissues may differ in various aspects, most previous studies on ECs have used normal cells. Here, we purified ECs from tumorous and normal human breast tissues, and studied the effect of radiation on angiogenesis and the relevant molecular mechanisms in these cells. We found that in normal tissue-derived endothelial cells (NECs), 4 Gy irradiation increased tube formation, MMP-2 expression and ERK pathway activation. In cancer-derived endothelial cells (CECs), however, 4 Gy irradiation significantly reduced tube formation, increased the production of angiostatin and interleukin-6 (IL-6), and up-regulated AKT and JNK pathway activation. Knockdown experiments showed that siMMP-2 efficiently inhibited tube formation by irradiated NECs, while siPlasminogen effectively attenuated the irradiation-induced suppression of tube formation and the up-regulation of angiostatin in CECs. Moreover, siIL-6 clearly inhibited the irradiation-induced generation of angiostatin in CECs. Inhibition of ERK with a pharmacological inhibitor or siRNAs markedly suppressed irradiation-induced tube formation and MMP-2 up-regulation in NECs, while the inhibition of either AKT or JNK with pharmacological inhibitor or siRNA treatment of CECs clearly attenuated the irradiation-induced inhibition of tube formation and the up-regulation of angiostatin and IL-6. These observations collectively demonstrate that there are distinct differences in the radiation responses of NECs and CECs, and might provide important clues for improving the efficacy of radiation therapy. Citation Format: Eun-Taex Oh, Moon-Taek Park, Min-Jeong Song, Hyemi Lee, Heon Joo Park. Radiation-induced angiogenic signaling pathway in endothelial cells isolated from normal and cancer tissue of human breast. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3905. doi:10.1158/1538-7445.AM2013-3905